Avian influenza virus (AIV) is a common disease in birds. The first infection of subtype H5N1 AIV among poultry was detected in 1996 in a farmed goose in Guangdong Province, China, and in humans a year later in Hong Kong [1]. Since then, H5N1 AIV has caused an outbreak of avian influenza that is spreading to many regions of the world. The affected areas to date include Europe, the Middle East and, particularly, Asia [2]. According to the latest reports from the World Health Organization (WHO), there have been a total of 328 confirmed human H5N1 avian influenza cases, 200 of which resulted in the death of the patient. Of the 328 cases, a significant majority (106 cases, 85 deaths) have been reported from Indonesia [3,4]. According to WHO, the world now is in phase 3 (of 6) of a pandemic alert based upon the evolution of the virus into a strain that is capable of efficient human to human transmission [5].
In June, 2006, 27 of 33 provinces in Indonesia had reported outbreaks of H5N1 in poultry, resulting in more than 16 million poultry deaths from sickness and culling [6]. The poultry industry has lost millions of dollars to avian influenza. This loss has affected the incomes of millions of people whose livelihoods depend on poultry. These outbreaks of HPAI (H5N1) in poultry, and now the increasing number of cases in humans, are a cause for concern. The ability to accurately and timely detect the presence of the pathogen in the initial stages of an outbreak will go a long way in controlling the disease. In addition, it can reduce indiscriminate use of antibiotics and provide the option of using antiviral therapy in a timely manner.
Various methods available for the diagnosis of influenza include virus isolation, detection of viral antigens by enzyme-linked immunoabsorbent assay (ELISA), molecular detection by RT-PCR and serological tests. Standard virus-isolation procedures have the disadvantage of requiring several days to obtain results, thereby making them of limited use to a clinician. The disadvantages of RT-PCR include the high costs involved, the need for technically proficient staff, likelihood of contamination and the consequent risk of false positive results. In addition, PCR primers may require constant updating because of antigenic drift [7]. Virus neutralization, hemagglutination inhibition (HI), ELISA and immunoblot test are preferred methods for serological diagnosis. However, neutralization assay and HI assay are not considered highly sensitive and necessitate further sub-typing and also are quite labor-intensive and time-consuming [8,9] and so are not ideal for large-scale routine testing of sera. ELISA has been widely used as a pre-screening tool for investigating large numbers of samples, but indirect ELISA systems are commercially available only for chicken and turkey sera due to the unavailability of species-specific secondary antibodies of other species. A further limitation of indirect ELISA is the need for high antigen purity. The most significant disadvantage of the indirect ELISA, however, is that the HA antigen is known to cross-react with the other subtypes of viruses. As a consequence, the indirect ELISA is not a dependable method for detection.
Most of these methods not only are cumbersome and labor intensive but also are time-consuming and include a risk of obtaining false positive results. A further limitation of these techniques is that influenza viruses are segmented genome RNA viruses which are known to undergo continuous mutations and genetic re-assortments (antigenic drift), making it difficult to detect the virus [10].
In view of the shortcomings of these conventional assays, and because of the risk that AIV infection poses to wildlife, domesticated animals and humans, there is a high need for a new assay which is rapid, easy to use and specific for the detection of the H5 subtype of AIV. The present invention represents a breakthrough in the diagnosis and surveillance of H5 subtype of AIV.